Transposable Mutagenesis for an E. coli Methionine or Histidine Synthesis Gene Jon Kissel Mary Maschek

Transposon mutagenesis Mutating prototrophic bacteria to become auxotrophic using a transposon Making plasmid library from bacterial genome Sequencing plasmid with interrupted gene

Transposons Mobile DNA elements Randomly insert into genome Can contain resistance genes

Types of transposons Size Number of genes

Bacteriophage Mu Lysogenically inserts into bacterial genome Inserts at multiple locations Can recombine out of genome and form a plasmid library

Plasmid Libraries Contain bacterial genome Individual genes are located on each plasmid Can be used to transform wild-type bacteria

Inserting the Transposon EZ::TN™ Tnp Transposome™ Kit Electroporation

EZ::TN™ Tnp Transposon Epicentre EZ::TN™ Tnp Transposome™ Kit protocol

Finding the Auxotroph Plate cells on minimal medium + met/his Replica plate on minimal medium Select for colonies that don’t grow

Making the Library DNA purification Restriction digest End-It DNA Repair Kit DNA ligation

Finding Resistance Mix library with wild-type E. coli Plate on kanamycin Select for kanamycin-resistance

Plasmid Purification Good ole Biology 107 Run on gel just to be sure

Sequencing and BLAST Send purified plasmid to IUPUI Primers come with transposome kit that tell sequencers where to begin BLAST returned sequence

Epicentre EZ::TN™ Tnp Transposome™ Kit

Timeline September – make plates, LB broth September – begin work with transposome and obtain auxotroph September 27-October 15 – ligate genomic DNA, plasmid library ready October – isolate kanamycin-resistant colony October – isolate and purify plasmid October 29-November 5 – enter sequence into BLAST November 5 to December 8 – work on manuscript

Budget EZ::TN™ Tnp Transposome™ Kit $375? End-It DNA Repair kit $70 Sequencing at IUPUI $17

Grade Agreement Obtaining auxotroph 15 points Purifying DNA 5 points Digesting DNA 5 points Assembly of plasmid library 10 points Obtaining kanamycin-resistant colony 10 points BLAST results give methionine or histidine synthesis gene 5 points 5 points